Mass spectrometry, particularly tandem mass spectrometry (MS/MS) is attractive for many applications due to its high selectivity, wide dynamic range, and high throughput capabilities. Analysis of isomers by mass spectrometry usually is complicated by similarity between their mass spectra, and often requires adequate chromatographic separation between compounds in order to eliminate mutual interference. Differentiation of isomers using mass spectrometry is presently achieved through derivatization, choice of collision gas, collision energy, or most commonly in conjunction with chromatographic separation of isomers. Each approach is usually compound specific, such as is described in the following references, all of which are hereby incorporated by reference: Borth, S.; Hansel, W.; Rosner, P.; Junge, T., “Regioisomeric differentiation of 2,3- and 3,4-methylenedioxy ring-substituted phenylalkylamines by gas chromatography/tandem mass spectrometry.” J Mass Spectrom. 2000, 35, 705; Vouros, P.; Muller, D. R.; Richter, W. J. “Low-energy collision-induced dissociation of B1-type sugar ions formed from peracetylated methyl pentosides and methyl 6-deoxyhexosides.” J. Mass Spectrom. 1999, 34, 346; Sheeley, D. M.; Reinhold, V. N. “Structural Characterization of Carbohydrate Sequence, Linkage, and Branching in a Quadrupole Ion Trap Mass Spectrometer: Neutral Oligosaccharides and N-Linked Glycans.” Anal. Chem. 1998, 70, 3053; Zimmermann, R.; Rohwer E. R.; Heger, H. “In-line catalytic derivatization method for selective detection of chlorinated aromatics with a hyphenated gas chromatography/laser mass spectrometry technique: A concept for comprehensive detection of isomeric ensembles.” Anal. Chem. 1999, 71, 4148–4153; Seymour, J. L.; Turechek, F. “Distinction and quantitation of leucine-isoleucine isomers and lysine-glutamine isobars by electrospray ionization tandem mass spectrometry (MS n, n 5 2, 3) of copper(II)-diimine complexes.” J. Mass Spectrom. 2000, 35, 566–571. It is common that the same mass ion fragments are present among all the isomers. This may create difficulties for analysis of isomers if they are present in a mixture at variable concentrations.
Additionally, structural isomers may be separately analyzed utilizing blackbody infrared radiative dissociation, as described in Schnier, P. D. W., E. R. “Analysis of isomeric mixtures using blackbody infrared radiative dissociation: Determining isomeric purity and obtaining individual tandem mass spectra simultaneously.” Anal. Chem 1998, 70, 3033–3041, hereby incorporated by reference; mass-analyzed ion kinetic energy spectroscopy/collision induced dissociation (CID) method, as described, for example, in Krishna, P.; Prabhakar, S.; Vairamani, M. “Differentiation of derivatized leucine and isoleucine by tandem mass spectrometry under liquid secondary ion mass spectral conditions.” Rapid Commuin Mass Spectrom. 1998, 12, 1429–1434, hereby incorporated by reference; or compound derivatization followed by low energy CID at conditions preferential for each of the isomers, as described, for example in, Desaire, H. L.; Leary J. A. “Multicomponent quantification of diastereomeric hexosamine monosaccharides using ion trap tandem mass spectrometry.” Anal. Chem 1999, 71, 4142–4147, hereby incorporated by reference.
Desaire et al. developed a method for relative quantitation of a mixture of unresolved isomers that is based on the assumption that the total signal of MS/MS transitions is a linear combination of signal intensities of product ions from multiple components. For calculations the method utilizes branching ratios of product ions of pure analytes, assumes equal sensitivity to all the analytes, and normalizes total concentration of all components of a mixture to 100%. Accordingly, the method does not allow obtaining absolute values of concentration and the result of the calculations is presented as percent of total concentration of all isomers present in a mixture.
Bennett et al., “Simultaneous analysis of butene isomer mixtures using process mass spectrometry.” J Am Soc. Mass Spectrom. 2000, 11, 1079–85, hereby incorporated by reference, proposed a method for analyzing mixture of isomers utilizing single MS analyzer for process mass spectrometry. The approach can be used to analyze a mixture of isomers within infrequently encountered molecular weight range. Simultaneous quantitation of isomer mixtures in complex samples within commonly encountered range of m/z, like in biological samples, is not feasible with single MS detection because of high potential for interference.